NK degranulation assays were performed as described previously using anti-CD107a-FITC (Cat. no. 555800, BD Biosciences, Oxford, UK, RRID:AB_396134 ) or isotype control-FITC (Cat no. 555748, BD Biosciences, RRID:AB_396090 ) and anti-CD3-PE-Cy7 (Cat. no. 737657, Beckman Coulter, High Wycombe, UK RRID:AB_2636813 ) and anti-CD56-PE (Cat. no. A07788, Beckman Coulter, RRID:AB_2636814 ) antibodies and PBMC derived from buffy coats or donor blood, except that infected HF-TERTs were harvested using HyQtase for 3 min at 37°C, instead of Trypsin/EDTA (Prod'homme et al., 2007 (link), 2010 (link); Fielding et al., 2014 (link)). Blocking experiments were performed as previously described except using B7-H6 blocking antibody (CH31) or isotype IgG1 control antibody at 10 µg/ml (Biolegend, Cat. no. 401404, Clone MG1-45, RRID:AB_345426 or Biolegend, Cat no. 401402 Clone MG1-45, RRID:AB_345424 ) (Fielding et al., 2014 (link)).
Clone mg1 45
Manufactured by BioLegend
The Clone MG1-45 is a lab equipment product manufactured by BioLegend. It is a cell surface marker that can be used for the identification and analysis of specific cell types in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd107a fitc, by BD (1 mentions)
Anti cd3 pe cy7, by Beckman Coulter (1 mentions)
Anti cd56 pe, by Beckman Coulter (1 mentions)
7 aad viability staining solution, by BioLegend (1 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
Falcon tube, by BD (1 mentions)
T75 flask, by Corning (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
4 protocols using clone mg1 45
1
NK Cell Degranulation Assays with Flow Cytometry
2
Cell Surface Marker Analysis by FACS
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Cells were washed with cold fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline (PBS) containing 1% sodium azide and 2% FBS) and incubated with the indicated mAbs at 4 °C for 30 min, after blocking with purified mouse IgG (BioLegend, clone MG1-45, 0.5 μg/mL, 4 °C, 15 min). After several washes, the cells were stained with 7-AAD Viability Staining Solution (BioLegend) and analyzed using a BD FACSAria III Cell Sorter (BD, Franklin Lakes, NJ, USA) and FlowJo software version 10 (BD).
3
Phagocytosis Assay for Tumor-Dendritic Cell Interactions
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Tumor cells were stained with 2 µM of the fluorescent probe 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE, Invitrogen, Thermo Fisher Scientific, catalog# C34554), according to manufacturer's instructions. Fluorescently–labeled (CFSE or GFP) tumor cells were seeded in T75 flasks (Corning), adhered and treated with OXP or CDDP. After treatment, tumor cells (5 × 105 cells, target) and DCs (5 × 105 cells, effector) were co-cultured at a 1:1 ratio in Falcon tubes (BD Falcon) in X-VIVO-15 supplemented with 2% HS (final concentration 1 × 106/mL). Co-cultures were stained with an APC-labeled primary antibody recognizing a specific DC surface marker (CD11c for CD1c+ and CD16+ DCs, CD123 for pDCs; see also Table S1) and analyzed by flow cytometry. Extent of phagocytosis was determined as the percentage of double positive events (i.e., CD11c+ or CD123+-APC/GFP+ or CFSE+). For blocking experiments, tumor cells were pre-incubated (30 min at 4°C) with blocking agents or negative controls: CRT blocking peptide (20 µg/mL, MBL International, catalog# JM-3077BP-50), irrelevant gp100 peptide272-300 (20 µg/mL, JPT), anti-CD91 (20 µg/mL, Thermo Fisher Scientific, clone 8G1, catalog# MA1-27198), or IgG1, k (20 µg/mL, Biolegend, clone MG1-45). Extra volume blocking agents or matched controls were added to co-cultures at same final concentration.
4
HCMV Modulation of NKp30-Mediated Cytotoxicity
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HF-TERTs were seeded into 96-well plates and either mock-infected or infected with HCMV or US12 family deletion mutants (MOI 10) for 72 hr. The CT299 reporter line (2B4 cells stably transfected with an NFAT-GFP reporter and NKp30) was washed in complete RPMI and 50,000 reporter cells added per well (5:1 ratio of reporter to target cells). In some experiments, the B7-H6 blocking antibody (CH31) or isotype IgG1 control antibody (Biolegend, Cat. no. 401404, Clone MG1-45, RRID:AB_345426 or Biolegend Cat no. 401402 Clone MG1-45, RRID:AB_345424 ) at 10 µg/ml were also added to the wells. After 24 hr incubation, reporters were harvested, washed in FACS buffer and fixed with 2% paraformaldehyde before analysis by flow cytometry.
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